In the gene of eukaryotes, there are many cases where genetic information stipulating the amino acid sequence of protein is interrupted. A moiety having the genetic information of amino acid sequence of protein is called exon while a moiety having no genetic information of amino acid sequence is called intron. After an mRNA precursor is formed by a transcription of genetic DNA, it is subjected to a splicing so that an intron moiety is cut off whereby mature mRNA is resulted.
It has not been clarified yet why such an intron moiety is present in eukaryotes. However, it has been presumed that, in many cases, one exon is coded as a specific domain (functioning region) of protein and, when new protein having the same function is needed during the process of development, necessary protein is able to be produced by a combination of different exons.
With regard to a splicing of the mRNA precursor before being subjected to the splicing, there has been also known the case where not only intron is cut off but also exon moiety is cut off to give mRNA coding for different protein having the function of the same type.
For example, calcitonin gene has six exons—A, B, C, D, calcitonin CCP and CGRP (calcitonin gene related peptide). Exon A and exon B are non-translated region while translated region is other four exons. When a transcription is carried out in nucleus of cell, all exons are included but a process of the splicing varies depending upon organs. For example, in thyroid C cells, exon of the sixth CGRP is also spliced and, as a result, protein of the translated product becomes a peptide comprising C-D-calcitonin CCP mainly exhibiting an action of reducing a serum Ca. In hypothalamic cells, exon of the fifth calcitonin CCP is also spliced and, as a result, protein of the translated product becomes a peptide comprising C-D-CGRP mainly playing a role of regulation of pain and autonomous activity.
When an exon moiety is divided into some as such, it is possible to produce different proteins where several exons are bonded if necessary. Although it has been explained to divide an exon for such a purpose, there has been almost no clarification yet for the necessity of intron except the preparation of an exon moiety. It has been known that many of introns have sequences of 5′-GT and AG-3′ at the terminals and that there is an intermediate region abundant in pyrimidine and it has been believed that a splicing is carried out by recognizing those sequences at both terminals.
Phospholipase A2 is widely distributed in mammals and microbes and it is mostly a membrane-bound enzyme and participates in metabolism of membrane phospholipids. A cytosolic phospholipase A2 (cPLA2α) of 85 kDa is a kind of phospholipase A2 and cuts out arachidonic acid mostly from membrane phospholipids producing physiologically active substances by arachidonic acid cascades such as prostaglandin, thromboxane, leukotriene, etc. derived from arachidonic acid. It has been also known that the liberated arachidonic acid participates in various nervous functions in the brain and, until now, the present inventors have shown by a northern blot technique and an in situ hybridization that cPLA2 α is abundantly expressed in cranial nerve cells.
On the other hand, kainic acid is a kind of amino acid and has been isolated as an anthelmintic component in Digenea simplex. Since kainic acid has a chemical structure similar to glutamic acid, it has been known as a substance binding to a glutamic acid receptor in the brain and the nerve cells of animals resulting in a neuron exciting action.
In order to check the function of phospholipase A2 in the brain, the present inventors have applied kainic acid stimulation or electric stimulation thereto and found a novel phospholipase A2 (455 amino acids; molecular weight: about 50 K) which transiently expresses being limited to dentate gyrus of hippocampus. This enzyme is a partial protein initiating from the 308th methionine of a cytosolic phospholipase A2a (85 K) and, since it also expresses in genetically defective mouse of the said enzyme, it contains a specific promoter which site-specifically expresses in response to stimulation. Although this enzyme is not present under a non-stimulated state, it is expressed by electric stimulation and kainic acid stimulation and, unlike the conventional phospholipase A2, it is independent upon calcium unlike the conventional phospholipase A2, produces eicosanoid, regulates a cerebral function and participates in denaturation, apoptosis and regeneration of nerve cells whereby it is believed to be a molecule holding the key to those cerebral functions.
Further, this novel phospholipase A2 (455 amino acids; molecular weight: about 50 K) is a partial protein initiating from the 308th methionine of a known cytosolic phospholipase A2a (85 K), a promoter region specific for expressing this protein is present in the intron moiety immediately before that and the present inventors have found that, in the intron, there is an intron having a function of making the initiation of transcription of RNA possible. Under a usual state, this intron has no function of initiating the transcription of RNA. However, when a certain condition is set, it has a function of initiating the transcription of RNA not from the inherent transcription position but from the moiety of the base sequence of this intron.